FIG 4.

Effects of the M¹⁵⁰ and R¹⁵⁴ residues of the C protein on binding with STAT1. (A and B) 293T cells were transfected with the indicated plasmids and solubilized in cell lysis buffer after 24 h. Cell lysates were immunoprecipitated with an anti-FLAG (A) or anti-HA (B) antibody together with protein G Sepharose, and the immunoprecipitates were analyzed by SDS-PAGE followed by Western blotting using an anti-HA (A) or anti-C (B) antibody. A part of the cell lysates was processed for SDS-PAGE and Western blotting to confirm protein expression using anti-HA, anti-FLAG, and anti-C antibodies. (C) Accumulated EGFP expressed from pISRE-EGFP or pGAS-EGFP reporter plasmid after addition of IFN-α or IFN-γ was shown by Western blotting in the presence of FL-Y3 protein mutants, as indicated in the figure. Expression of FL-Y3 protein mutants in one experiment was also shown. (D and E) The experiments were repeated at least three times, and band signals of accumulated EGFP were quantified with the MultiGauge software. The average values were plotted on the graph, and the error bar indicates the standard deviation. The P value was calculated on the basis of the Mann-Whitney U test.